Pulmonary hypertension biomarker

ABSTRACT

Pulmonary hypertension is a progressive disease of various origins that is associated with vascular remodelling and results in right heart dysfunction. Accumulating evidence indicates important roles of immune cells and inflammatory chemokines in the pathogenesis and progression of pulmonary hypertension. We have identified CCL21 as anti-remodelling efficacy biomarker for pulmonary hypertension. CCL21 was found to be highly sensitive and specific in discriminating pulmonary hypertension patients from matched controls. CCL21 was upregulated in pulmonary hypertension and down-regulated with treatment with an anti-remodelling agent.

FIELD OF THE INVENTION

The invention is in the field of biomarkers in respiratory disease. Inparticular, it relates to the use of CCL21 expression as a biomarker forpulmonary hypertension.

BACKGROUND OF THE INVENTION

Pulmonary hypertension is a progressive disease of various origins thatis associated with a poor prognosis and results in right heartdysfunction. In all its variant presentations, this disease is estimatedto affect up to 100 million people worldwide¹. According to the currentclassification of pulmonary hypertension, which was agreed upon at the4th World Symposium on Pulmonary Hypertension in 2008, five categoriesof chronic pulmonary hypertension exist.

Pulmonary hypertension (PH) is defined as a mean rise in pulmonaryarterial pressure >25 mmHg at rest (>30 mmHg following exercise). Group1 PH can be further subdivided into diseases where increased pulmonaryvascular resistance is due to pre-capillary micro-angiopathy (diagnosedas a pre-capillary wedge pressure <15 mmHg). Within this group we findidiopathic pulmonary arterial hypertension (IPAH) and familial pulmonaryarterial hypertension, associated pulmonary arterial hypertension,pulmonary arterial hypertension with venous/capillary involvement, &persistent pulmonary hypertension of the newborn. Group 2 includespulmonary hypertension due to left heart diseases whereas Group 3includes pulmonary hypertension associated with lung disease/hypoxemia(e.g. COPD) and Group 4 pulmonary hypertension associated with chronicthromboembolic disorders².

Despite advances in understanding of the underlying pathobiology ofpulmonary hypertension and some improvements in diagnosis anddevelopment of novel therapeutics, there is still significant unmetmedical need and unacceptable rates of morbidity and mortality acrossthe spectrum of pulmonary hypertension.

The subcategories of pulmonary hypertension differ in their underlyingcauses. However, they all are characterized by excessive pulmonaryvasoconstriction and abnormal vascular remodelling unique plexiformlesions. Endothelial dysfunction associated with inflammation andoxidative stress and vascular smooth muscle cell (SMC) proliferation areprominent features of pulmonary arterial hypertension³⁻⁵. Thesestructural changes suggest a switch from a quiescent state to aproliferative, apoptosis-resistant cellular phenotype^(6,7). Vascularremodelling leads to a chronic elevation of pulmonary vascularresistance, right heart failure and death.

Several studies have also suggested a role for immune mechanisms inpulmonary arterial hypertension pathophysiology⁸. Inflammatory cells andintense chemokine production have been detected within remodeledpulmonary arteries, and vascular stromal cells have been shown to besensitive to inflammatory stimuli⁹. In addition, elevated circulatingcytokine levels have been measured in IPAH^(10,11). Up to one-third ofpatients with pulmonary arterial hypertension have circulatingautoantibodies against various vascular self-antigens^(12,13). Thissuggests that the adaptive immune system, consisting of T and Blymphocytes, is involved, and indeed perivascular T and B lymphocyteshave been detected in pulmonary vascular pulmonary arterial hypertensionlesions¹⁴. Work on chronic inflammatory disorders and autoimmunediseases suggests that pathogenic antibodies and T cells may also begenerated locally, in the targeted organ, in highly organized ectopiclymphoid follicles commonly called tertiary lymphoid tissues (tLTs)¹⁵.The role of tLTs in chronic pulmonary diseases is gaining in importance,especially in chronic obstructive pulmonary diseases¹⁶, in idiopathicpulmonary fibrosis¹⁷, and in obliterative bronchiolitis¹⁸ and morerecently pulmonary arterial hypertension¹⁹. Ectopic formation ofsecondary lymphoid tissue is initiated by the local attraction of naiveT and B cells. Hence, the local production of homeostatic chemokines,and lymphocyte survival factors such as CCL21, attractingCCR7-expressing cells, such as mature DCs, naive T cells, and B cells²⁰,may be a critical event in the formation of ectopic lymphoid tissue.CCL21 expression, in particular, has been recently detected in tLTs inexplanted lungs from patients with IPAH¹⁹.

Current guidelines recommend the use of either brain natriuretic peptide(BNP) or the N-terminal fragment of pro-BNP (NT-proBNP) as biomarkersfor mortality risk stratification. Natriuretic peptides were the firstblood-derived markers of pulmonary hypertension. Nagaya et al were thefirst to show that plasma levels of BNP have a prognostic significancein pulmonary hypertension²¹. BNP levels predicted mortality in adultpatients with symptomatic congenital heart disease²², and BNP was alsoan independent predictor of therapy response. In a retrospective studyin patients with pulmonary arterial hypertension, serial measurements ofNT-proBNP (a by-product of BNP synthesis) were associated withsurvival²³. Log-transformation of NT-proBNP values identified patientswith pulmonary arterial hypertension who were at risk of adverse eventswith a specificity of 98% and a sensitivity of 60%²⁴.

However, BNP or NT-proBNP, are markers of myocardial strain, excessivestretching of the heart, and increased heart rate and do not directlyreflect changes in distal pulmonary arteries in the lung, which areresponsible for driving pulmonary hypertension pathophysiology.Remodelling changes in the heart and right ventricle specifically, arethought to follow pulmonary arteries remodelling. Thus, it is of crucialinterest to assess and monitor pulmonary artery remodelling usingsurrogate non-invasive circulating biomarkers before the effect can bevisualised in the right heart as a result of disease progression.

Biomarkers that specifically indicate the pathologic mechanism, theseverity of the disease or the treatment response would be ideal toolsfor the management of pulmonary hypertension and would also facilitatethe successful execution of future clinical trials.

SUMMARY OF THE INVENTION

It has now been found that CCL21 is a highly specific and sensitivebiomarker for discriminating pulmonary hypertension patients frommatched controls.

The invention therefore provides for a method for determining if asubject has pulmonary hypertension, comprising

-   -   a) providing a biological sample obtained from a subject        suspected of having pulmonary hypertension;    -   b) assaying the biological sample for the level of CCL21        expression and/or CCL21 protein; and    -   c) comparing the amount of CCL21 expression and/or of CCL21        protein to a baseline value that is indicative of the amount of        CCL21 expression and/or of CCL21 protein in a subject that does        not have pulmonary hypertension;        wherein a statistical significant increased amount of CCL21        expression and/or of CCL21 protein compared to the baseline        value is indicative of pulmonary hypertension.

The invention also provides for a method of treating a patient havingpulmonary hypertension, comprising

-   -   a) assaying a biological sample obtained from the patient for        the level of CCL21 expression and/or CCL21 protein; and    -   b) administering a therapeutically effective amount of a        pulmonary hypertension antagonist if the patient has a        statistical significant increased amount of CCL21 expression        and/or CCL21 protein compared to the amount of CCL21 expression        and/or of CCL21 protein to a baseline value that is indicative        of the amount of CCL21 expression and/or of CCL21 protein in a        subject that does not have pulmonary hypertension.

The invention also provides for a method of predicting the likelihoodthat a patient having pulmonary hypertension will respond to treatmentwith a pulmonary hypertension antagonist, comprising, assaying abiological sample obtained from the patient for the level of CCL21expression and/or CCL21 protein; and wherein an increased level of CCL21expression and/or CCL21 protein relative to a baseline value isindicative of an increased likelihood that the patient will respond totreatment with the pulmonary hypertension antagonist.

In some embodiments of these aspects, the step of assaying comprisesassaying the biological sample for a nucleic acid sequence of CCL21expression, such as a nucleic acid is selected from CCL21 ribonucleicacid (RNA) or a fragment thereof and complementary deoxyribonucleic acid(cDNA) or a fragment thereof. In some other embodiments t step ofassaying comprises assaying the biological sample for a CCL21 protein orfragment thereof.

In some other embodiments of these aspects the biological sample isselected from blood, serum, plasma, urine, saliva, faeces and a tissuesample.

In some other embodiments of these aspects the step of assayingcomprises a technique selected from Northern blot analysis, polymerasechain reaction (PCR), reverse transcription-polymerase chain reaction(RT-PCR), TaqMan-based assays, direct sequencing, dynamicallele-specific hybridization, primer extension assays, oligonucleotideligase assays, temperature gradient gel electrophoresis (TGGE),denaturing high performance liquid chromatography, high-resolutionmelting analysis, DNA mismatch-binding protein assays, capillaryelectrophoresis, Southern Blot, immunoassays, immunohistochemistry,ELISA, flow cytometry, Western blot, HPLC, and mass spectrometry.

The invention also provides a kit for use in determining if a subjecthas pulmonary hypertension predicting or for use in predicting thelikelihood that a patient having pulmonary hypertension will respond totreatment with a pulmonary hypertension antagonist, the kit comprising,

-   -   a) at least one probe capable of detecting the presence of CCL21        expression and/or CCL21 protein; and    -   b) instruction for using the probe to assay a biological sample        from the patient for the presence of CCL21 expression and/or        CCL21 protein.

In some embodiments of this aspect, the probe is selected from anoligonucleotide that specifically hybridizes to a region of a nucleicacid sequence of CCL21 expression or binding molecule capable of bindinga CCL21 protein or a fragment thereof.

In some embodiments of this aspect, the binding molecule is an antibodyor a fragment thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Candidate biomarker from lung mRNA expression profiles in thehypoxia/sugen rat model of PH following treatment with Imatinib.

FIG. 2: Human CCL21 protein levels in serum and plasma samples from PHpatients and matched controls (age, ethnicity, gender ratio-matched).

FIG. 3: CCL21 protein expression and localisation byimmunohistochemistry in human lung sections from PH patients undergoinglung transplantation.

DETAILED DESCRIPTION OF THE INVENTION Definitions

For purposes of interpreting this specification, the followingdefinitions will apply and whenever appropriate, terms used in thesingular will also include the plural and vice versa. Additionaldefinitions are set forth throughout the detailed description.

The term “CCL21” refers to human CCL21, unless it is specifiedotherwise, having amino acid sequence for example as defined inENST00000259607 (Ensembl).

The term “CCL21” refers to the human CCL21 gene, unless it is specifiedotherwise, having nucleotide sequence for example as defined inENSP00000259607 (Ensembl).

The term “CCL21” is synonym to SCYA21; ECL; SLC; CKb9; TCA4; 6Ckine;6Ckine; exodus-2; “chemokine (C-C motif) ligand 21 [Homo sapiens(human)]”; “C-C motif chemokine 21”; “beta chemokine exodus-2”;“Efficient Chemoattractant for Lymphocytes”; exodus-2; “secondarylymphoid tissue chemokine”; “small inducible cytokine subfamily A(Cys-Cys), member 21”

As used herein, the term “gene” means the gene and all currently knownvariants thereof.

As used herein, the term “level” refers to RNA and/or DNA and/or proteincopy number of a biomarker according to the present invention.Typically, the level of a biomarker in a biological sample obtained froma patient under therapy is different (i.e. increased or decreased) fromthe level of the same biomarker in a similar sample obtained from ahealthy subject.

The terms “assaying”, “to assay”, “detection”, “detecting” and “todetect” refer to identifying the presence or absence of one or morebiomarker(s). The terms “measurement”, “measuring” and “to measure”refer to identify the presence, the absence or amount of one or morebiomarker(s).

As used herein, a “baseline value” generally refers to the level(amount) of CCL21 expression (e.g. mRNA) or CCL21 polypeptide (orprotein) in a comparable sample (e.g., from the same type of tissue asthe tested tissue), from a “normal” healthy subject that does notexhibit pulmonary hypertension. If desired, a pool or population of thesame tissues from normal subjects can be used, and the baseline valuecan be an average or mean of the measurements. Suitable baseline valuescan be determined by those of skill in the art without undueexperimentation. Suitable baseline values may be available in a databasecompiled from the values and/or may be determined based on publisheddata or on retrospective studies of patients' tissues, and otherinformation as would be apparent to a person of ordinary skillimplementing a method of the invention. Suitable baseline values may beselected using statistical tools that provide an appropriate confidenceinterval so that measured levels that fall outside the standard valuecan be accepted as being aberrant from a diagnostic perspective, andpredictive of pulmonary hypertension.

A “significant” increase in a value, as used herein, can refer to adifference which is reproducible or statistically significant, asdetermined using statistical methods that are appropriate and well-knownin the art, generally with a probability value of less than five percentchance of the change being due to random variation. In general, astatistically significant value is at least two standard deviations fromthe value in a “normal” healthy control subject. Suitable statisticaltests will be evident to a skilled worker. For example, a significantincrease in the amount of a protein compared to a baseline value can beabout 50%, 2-fold, or higher.

As used herein the terms “homolog” or “homologous” refers to apolynucleotide or polypeptide variant sharing common evolutionaryancestor or having at least 50% sequence identity with the wild type.

The term “binding molecule” as used herein means any protein or peptidethat binds specifically to CCL21 polypeptide. “Binding molecule”includes, but it is not limited to, antibodies and fragments thereof,such as immunologically functional fragments. The term “immunologicallyfunctional fragment” of an antibody or immunoglobulin chain as usedherein is a species of binding protein comprising a portion, regardlessof how that portion is obtained or synthesized of an antibody (anantigen-binding portion) that lacks at least some of the amino acidspresent in a full-length chain but which is still capable ofspecifically binding CCL21.

The term “antibody” refers to an intact immunoglobulin or a functionalfragment thereof. As used herein, the term “antibody” means apolypeptide comprising a framework region from an immunoglobulin gene orfragments thereof that specifically binds and recognizes an epitope,e.g. an epitope found on human CCL21. The term “antibody” includes wholeantibodies (such as monoclonal, chimeric, humanised and humanantibodies), including single-chain whole antibodies, andantigen-binding fragments thereof. The term “antibody” includesantigen-binding antibody fragments, single-chain antibodies, which cancomprise the variable regions alone, or in combination, with all or partof the following polypeptide elements: hinge region, CH1, CH2, and CH3domains of an antibody molecule.

As used herein, a binding molecule “capable of binding CCL21” isintended to refer to a binding molecule that binds to CCL21 with a K_(D)of a 1×10⁻⁶ M or less, or 1×10⁻⁷ M or less, or 1×10⁻⁸ M or less, or1×10⁻⁶ M or less, 1×10⁻¹⁰ M or less.

As used herein, the term “subject” includes any human or non-humananimal.

The term “non-human animal” includes all vertebrates, e.g., mammals andnon-mammals, such as non-human primates, sheep, dogs, cats, horses,cows, chickens, amphibians, reptiles, etc.

The term “patient” includes any human or non-human animal.

The term “pulmonary hypertension antagonist” means any molecule whichinhibits treat, prevent, cure pulmonary hypertension.

The term “treat”, “treating”, “treatment”, “prevent”, “preventing” or“prevention” includes therapeutic treatments, prophylactic treatmentsand applications in which one reduces the risk that a subject willdevelop a disorder or other risk factor. Treatment and/or prevention donot require the complete curing of a disorder and encompasses thereduction of the symptoms or underlying risk factors or at least aslowing down of the progression of the disease.

The term “comprising” means “including” as well as “consisting” e.g., acomposition “comprising” X may consist exclusively of X or may includesomething additional e.g., X+Y.

The term “about” in relation to a numerical value x means, for example,x+10%. References to a percentage sequence identity between two aminoacid sequences means that, when aligned, that percentage of amino acidsare the same in comparing the two sequences. This alignment and thepercent homology or sequence identity can be determined using softwareprograms known in the art, for example those described in section 7.7.18of Current Protocols in Molecular Biology (F. M. Ausubel et al., eds.,1987) Supplement 30. A preferred alignment is determined by theSmith-Waterman homology search algorithm using an affine gap search witha gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrixof 62. The Smith-Waterman homology search algorithm is disclosed inSmith & Waterman (1981) Adv. Appl. Math. 2: 482-489

CCL21 as Biomarker for Pulmonary Hypertension

Chemokine (C-C motif) ligand 21 (CCL21) is a small cytokine belonging tothe CC chemokine family. CCL21 is one of several CC cytokine genesinvolved in immunoregulatory and inflammatory processes. The CCcytokines are proteins characterized by two adjacent cysteines. Similarto other chemokines the protein encoded by this gene inhibitshemopoiesis and stimulates chemotaxis. This protein is chemotactic invitro for thymocytes and T cells and particularly naive T-cells, but notfor B cells, macrophages, or neutrophils. It is a high affinityfunctional ligand for chemokine receptor 7 that is expressed on T and Blymphocytes²⁵. CCL21 is thought to play a role in mediating homing oflymphocytes to secondary lymphoid organs. More recently, CCL21expression has been detected in ectopic formation of secondary lymphoidtissue in tLTs in explanted lungs from patients with idiopathicpulmonary arterial hypertension¹⁹. In this study, CCL21 was studied inboth human and rat samples. The rat and human CCL21 protein sequencesare 67% identical which indicates a high degree of homology between thetwo species.

Methods of Diagnosis and Treatment

The present invention provides for a method for determining if a subjecthas pulmonary hypertension, comprising

-   -   a) providing a biological sample obtained from a subject        suspected of having pulmonary hypertension;    -   b) assaying the biological sample for the level of CCL21        expression and/or CCL21 protein; and    -   c) comparing the amount of CCL21 expression and/or of CCL21        protein to a baseline value that is indicative of the amount of        CCL21 expression and/or of CCL21 protein in a subject that does        not have pulmonary hypertension;        wherein a statistical significant increased amount of CCL21        expression and/or of CCL21 protein compared to the baseline        value is indicative of pulmonary hypertension.

The present invention also provides for a method of predicting thelikelihood that a patient having pulmonary hypertension will respond totreatment with a pulmonary hypertension antagonist, comprising, assayinga biological sample obtained from the patient for the level of CCL21expression and/or CCL21 protein; and wherein an increased level of CCL21expression and/or CCL21 protein relative to a baseline value isindicative of an increased likelihood that the patient will respond totreatment with the pulmonary hypertension antagonist.

Furthermore, the present invention provides for a method of treating apatient having pulmonary hypertension, comprising

-   -   a) assaying a biological sample obtained from the patient for        the level of CCL21 expression and/or CCL21 protein; and    -   b) administering a therapeutically effective amount of a        pulmonary hypertension antagonist if the patient has a        statistical significant increased amount of CCL21 expression        and/or CCL21 protein compared to the amount of CCL21 expression        and/or of CCL21 protein to a baseline value that is indicative        of the amount of CCL21 expression and/or of CCL21 protein in a        subject that does not have pulmonary hypertension.

In embodiments of the present invention, the step of assaying comprisesassaying the biological sample for a nucleic acid of CCL21 expression

The result of CCL21 gene expression may be a polynucleotide (or nucleicacid). A polynucleotide or nucleic acid is a molecule comprising a chainof at least two nucleic acid monomers which can be deoxyribonucleoside,ribonucleosides and any modified nucleoside thereof. Specificallyincluded are DNA molecules as well as genomic and cDNA sequences, RNAmolecules such as mRNA and unspliced or partly spliced transcripts andsplicing products.

In one embodiment, the method for determining if a subject has pulmonaryhypertension, the method comprises

-   -   a) providing a biological sample obtained from a subject        suspected of having pulmonary hypertension;    -   b) assaying the biological sample for the level of CCL21        expression and/or CCL21 protein; and    -   c) comparing the amount of CCL21 expression and/or of CCL21        protein to a baseline value that is indicative of the amount of        CCL21 expression and/or of CCL21 protein in a subject that does        not have pulmonary hypertension; wherein a statistical        significant increased amount of CCL21 expression and/or of CCL21        protein compared to the baseline value is indicative of        pulmonary hypertension; wherein the step of assaying comprises        assaying the biological sample for a nucleic acid sequence of        CCL21 expression and wherein the nucleic acid is selected from        ribonucleic acid (RNA) or a fragment thereof and complementary        deoxyribonucleic acid (cDNA) or a fragment thereof. Preferably        the nucleic acid is cDNA amplified from CCL21 mRNA.

In another embodiment, the method of predicting the likelihood that apatient having pulmonary arterial hypertension will respond to treatmentwith a pulmonary arterial hypertension antagonist, comprising, assayinga biological sample obtained from the patient for the level of CCL21expression and/or CCL21 protein; and wherein an increased level of CCL21expression and/or CCL21 protein relative to a baseline value isindicative of an increased likelihood that the patient will respond totreatment with the pulmonary hypertension antagonist; wherein the stepof assaying comprises assaying the biological sample for a nucleic acidsequence of CCL21 expression and wherein the nucleic acid is selectedfrom ribonucleic acid (RNA) or a fragment thereof and complementarydeoxyribonucleic acid (cDNA) or a fragment thereof. Preferably thenucleic acid is cDNA amplified from CCL21 mRNA.

In yet another embodiment, the method of treating a patient havingpulmonary hypertension, comprising

-   -   a) assaying a biological sample obtained from the patient for        the level of CCL21 expression and/or CCL21 protein; and    -   b) administering a therapeutically effective amount of a        pulmonary hypertension antagonist if the patient has a        statistical significant increased amount of CCL21 expression        and/or CCL21 protein compared to the amount of CCL21 expression        and/or of CCL21 protein to a baseline value that is indicative        of the amount of CCL21 expression and/or of CCL21 protein in a        subject that does not have pulmonary hypertension; wherein the        step of assaying comprises assaying the biological sample for a        nucleic acid sequence of CCL21 expression and wherein the        nucleic acid is selected from ribonucleic acid (RNA) or a        fragment thereof and complementary deoxyribonucleic acid (cDNA)        or a fragment thereof. Preferably the nucleic acid is cDNA        amplified from CCL21 mRNA.

In other embodiments of the present invention, the step of assayingcomprises assaying the biological sample for a CCL21 protein or fragmentthereof.

CCL21 protein (or polypeptide) according to the present inventioncomprise the polypeptide obtained by (complete or incomplete)transcription and translation of the human CCL21 gene.

Polypeptide variants are also included in the present invention. Avariant polypeptide includes a molecule containing one or moredeletions, insertions and/or substitutions compared to the wild typepolypeptides obtained by transcription and translation of the wild typehuman CCL21 gene or by translation of the wild type polyribonucleotidetranscripts of that gene.

The biomarker according to the present invention may be a fragment or adegradation product of CCL21 polypeptide (or protein).

In one embodiment, the method for determining if a subject has pulmonaryhypertension, the method comprises

-   -   a) providing a biological sample obtained from a subject        suspected of having pulmonary hypertension;    -   b) assaying the biological sample for the level of CCL21        expression and/or CCL21 protein; and    -   c) comparing the amount of CCL21 expression and/or of CCL21        protein to a baseline value that is indicative of the amount of        CCL21 expression and/or of CCL21 protein in a subject that does        not have pulmonary hypertension; wherein a statistical        significant increased amount of CCL21 expression and/or of CCL21        protein compared to the baseline value is indicative of        pulmonary hypertension; and wherein the step of assaying        comprises assaying the biological sample for a CCL21 protein or        fragment thereof.

In another embodiment, the method for determining if a subject haspulmonary hypertension, the method comprises

-   -   a) providing a biological sample obtained from a subject        suspected of having pulmonary hypertension;    -   b) assaying the biological sample for the level of CCL21        expression and/or CCL21 protein; and    -   c) comparing the amount of CCL21 expression and/or of CCL21        protein to a baseline value that is indicative of the amount of        CCL21 expression and/or of CCL21 protein in a subject that does        not have pulmonary hypertension; wherein a statistical        significant increased amount of CCL21 expression and/or of CCL21        protein compared to the baseline value is indicative of        pulmonary hypertension; wherein the step of assaying comprises        assaying the biological sample for a CCL21 protein or fragment        thereof; and wherein the pulmonary hypertension is idiopathic        pulmonary arterial hypertension.

In another embodiment, the method of predicting the likelihood that apatient having pulmonary hypertension will respond to treatment with apulmonary hypertension antagonist, comprising, assaying a biologicalsample obtained from the patient for the level of CCL21 expressionand/or CCL21 protein; and wherein an increased level of CCL21 expressionand/or CCL21 protein relative to a baseline value is indicative of anincreased likelihood that the patient will respond to treatment with thepulmonary hypertension antagonist; and wherein the step of assayingcomprises assaying the biological sample for a CCL21 protein or fragmentthereof.

In another embodiment, the method of predicting the likelihood that apatient having pulmonary hypertension will respond to treatment with apulmonary hypertension antagonist, comprising, assaying a biologicalsample obtained from the patient for the level of CCL21 expressionand/or CCL21 protein; and wherein an increased level of CCL21 expressionand/or CCL21 protein relative to a baseline value is indicative of anincreased likelihood that the patient will respond to treatment with thepulmonary hypertension antagonist; wherein the step of assayingcomprises assaying the biological sample for a CCL21 protein or fragmentthereof; and wherein the pulmonary hypertension is idiopathic pulmonaryarterial hypertension.

In yet another embodiment, the method of treating a patient havingpulmonary hypertension, comprising

-   -   a) assaying a biological sample obtained from the patient for        the level of CCL21 expression and/or CCL21 protein; and    -   b) administering a therapeutically effective amount of a        pulmonary hypertension antagonist if the patient has a        statistical significant increased amount of CCL21 expression        and/or CCL21 protein compared to the amount of CCL21 expression        and/or of CCL21 protein to a baseline value that is indicative        of the amount of CCL21 expression and/or of CCL21 protein in a        subject that does not have pulmonary hypertension; and wherein        the step of assaying comprises assaying the biological sample        for a CCL21 protein or fragment thereof.

In another embodiment, the method of treating a patient having pulmonaryhypertension, comprising

-   -   a) assaying a biological sample obtained from the patient for        the level of CCL21 expression and/or CCL21 protein; and    -   b) administering a therapeutically effective amount of a        pulmonary hypertension antagonist if the patient has a        statistical significant increased amount of CCL21 expression        and/or CCL21 protein compared to the amount of CCL21 expression        and/or of CCL21 protein to a baseline value that is indicative        of the amount of CCL21 expression and/or of CCL21 protein in a        subject that does not have pulmonary hypertension; and wherein        the step of assaying comprises assaying the biological sample        for a CCL21 protein or fragment thereof; and wherein the        pulmonary hypertension is idiopathic pulmonary arterial        hypertension.

In other embodiments of the present invention, the step of assayingcomprises assaying the biological sample for a modified nucleic acidsequence of CCL21 expression or for a modified CCL21 protein or fragmentthereof.

Modifications of polynucleotides or polypeptides are well-known in theart. The modifications may be performed on one or more nucleosides oramino acid residues of the polynucleotides or polypeptides,respectively. Alternatively, or in combination with the afore-mentionedchemical modifications, the link between monomers may be modified.Further known modifications include the conjugation of tags or labels tothe polynucleotide or polypeptide biomarker.

Chemical modifications of polynucleotides, include, but are not limitedto, replacement of hydrogen by an alkyl, acyl or amino group, alterationof sugar moieties or inter-sugar linkages (i.e. phosphorothioate),labeling of nucleotides with radio-nucleotides (i.e. ³²P), conjugationwith tags or labeling molecules such as fluorescent tags (i.e.rhodamine, fluorescein, Cy3 and/or Cy5, chemiluminescent tags,chromogenic tags or other labels (i.e. digoxigenin or biotin andmagnetic particles). Modification of the sugar moieties, purine andpyrimidine heterocycles as well as heterocyclic analogues and tautomersthereof are also included herein. Illustrative examples arediaminopurine 8-oxo-N⁶-methyladenine, 7-deazaxanthine, 7-deazaguanine,N⁴,N⁴-ethanocytosin, N⁶,N⁶-ethano-2,6-diaminopurine, 5-methylcytosine,5-(C³-C⁶)-alkynylcytosine, 5-fluorouracil, 5bromouracil,2-hydroxy-5methyl-4-triazolopyridin, isocytosin, isoguanin, inosine andthe examples described in U.S. Pat. No. 5,432,272; Scheit, NucleotideAnalogs, John Wiley, New York, 1980; Freier and Altmann, Nucl. AcidRes., 1997, 25(22), 4429-43; Toulme', J. J., Nature Biotechnology19:17-18 (2001); Manoharan M.; Biochemica et Biophysica Acta1489:117-139 (1999); Freier S. M., Nucleic acid Research, 25:4429-4443(1997); Uhlman E., Drug Discovery & Development, 3: 203-213 (2000);Herdewin P., Antisense & Nucleic acid Drug Dev., 10:297-310 (2000).

A wide variety of labeling and conjugation techniques are known by thoseskilled in the art. Polynucleotides or nucleic acids labeling can beachieved for example by oligo-labeling, nick translation, end-labelingor PCR amplification using a labeled primer.

The chemical modifications of a polynucleotide biomarker according tothe present invention preferably comprise radioisotope labeling and/orfluorescent agent labeling. More preferably, the polynucleotidebiomarker(s) according to the present invention, especially whenamplified in number copies by polymerase chain reaction (PCR), comprisesa fluorescent tag (e.g. TaqMan® Gene Expression Assays consist of a pairof unlabeled PCR primers and a TaqMan® probe with a FAM™ or VIC® dyelabel on the 5′ end, and minor groove binder (MGB) nonfluorescentquencher (NFQ) on the 3′ end.

Biological Samples

In embodiments of the present invention, the biological sample isselected from blood, serum, plasma, urine, saliva, feces and a tissuesample.

A sample which is “provided” can be obtained by the person (or machine)conducting the assay, or it can have been obtained by another, andtransferred to the person (or machine) carrying out the assay.

Many suitable sample types will be evident to a skilled worker. In oneembodiment of the invention, the sample is a blood sample, such as wholeblood, plasma, or serum (plasma from which clotting factors have beenremoved). For example, peripheral or venous plasma or serum can be used.In another embodiment, the sample is urine, sweat, or another body fluidinto which proteins are sometimes removed from the blood stream. In thecase of urine, for example, the protein is likely to be broken down, sodiagnostic fragments of the proteins of the invention can be screenedfor. In another embodiment, the sample is pulmonary tissue, which isharvested, e.g., after a biopsys. Methods for obtaining samples andpreparing them for analysis are conventional and well-known in the art.

In one embodiment, the method for determining if a subject has pulmonaryhypertension, the method comprises

-   -   a) providing a biological sample obtained from a subject        suspected of having pulmonary hypertension;    -   b) assaying the biological sample for the level of CCL21        expression and/or CCL21 protein; and    -   c) comparing the amount of CCL21 expression and/or of CCL21        protein to a baseline value that is indicative of the amount of        CCL21 expression and/or of CCL21 protein in a subject that does        not have pulmonary hypertension;    -   wherein a statistical significant increased amount of CCL21        expression and/or of CCL21 protein compared to the baseline        value is indicative of pulmonary hypertension; wherein the step        of assaying comprises assaying the biological sample for a        nucleic acid sequence of CCL21 expression; wherein the nucleic        acid is selected from ribonucleic acid (RNA) or a fragment        thereof and complementary deoxyribonucleic acid (cDNA) or a        fragment thereof; and wherein the biological sample is selected        from blood or plasma or serum. Preferably the nucleic acid is        cDNA amplified from CCL21 mRNA.

In another embodiment, the method of predicting the likelihood that apatient having pulmonary hypertension will respond to treatment with apulmonary hypertension antagonist, comprising, assaying a biologicalsample obtained from the patient for the level of CCL21 expressionand/or CCL21 protein; and wherein an increased level of CCL21 expressionand/or CCL21 protein relative to a baseline value is indicative of anincreased likelihood that the patient will respond to treatment with thepulmonary hypertension antagonist; wherein the step of assayingcomprises assaying the biological sample for a nucleic acid sequence ofCCL21 expression; wherein the nucleic acid is selected from ribonucleicacid (RNA) or a fragment thereof and complementary deoxyribonucleic acid(cDNA) or a fragment thereof and wherein the biological sample isselected from blood or plasma or serum. Preferably the nucleic acid iscDNA amplified from CCL21 mRNA.

In yet another embodiment, the method of treating a patient havingpulmonary hypertension, comprising

-   -   a) assaying a biological sample obtained from the patient for        the level of CCL21 expression and/or CCL21 protein; and    -   b) administering a therapeutically effective amount of a        pulmonary hypertension antagonist if the patient has a        statistical significant increased amount of CCL21 expression        and/or CCL21 protein compared to the amount of CCL21 expression        and/or of CCL21 protein to a baseline value that is indicative        of the amount of CCL21 expression and/or of CCL21 protein in a        subject that does not have pulmonary hypertension; wherein the        step of assaying comprises assaying the biological sample for a        nucleic acid sequence of CCL21 expression; wherein the nucleic        acid is selected from ribonucleic acid (RNA) or a fragment        thereof and CCL21 ribonucleic acid (RNA) complementary        deoxyribonucleic acid (cDNA) or a fragment thereof and wherein        the biological sample is selected from blood or plasma or serum.        Preferably the nucleic acid is cDNA amplified from CCL21 mRNA.

In another embodiment, the method for determining if a subject haspulmonary hypertension, the method comprises

-   -   a) providing a biological sample obtained from a subject        suspected of having pulmonary hypertension;    -   b) assaying the biological sample for the level of CCL21        expression and/or CCL21 protein; and    -   c) comparing the amount of CCL21 expression and/or of CCL21        protein to a baseline value that is indicative of the amount of        CCL21 expression and/or of CCL21 protein in a subject that does        not have pulmonary hypertension; wherein a statistical        significant increased amount of CCL21 expression and/or of CCL21        protein compared to the baseline value is indicative of        pulmonary hypertension; wherein the step of assaying comprises        assaying the biological sample for a CCL21 protein or fragment        thereof and wherein the biological sample is selected from blood        or plasma or serum.

In another embodiment, the method of predicting the likelihood that apatient having pulmonary hypertension will respond to treatment with apulmonary hypertension antagonist, comprising, assaying a biologicalsample obtained from the patient for the level of CCL21 expressionand/or CCL21 protein; and wherein an increased level of CCL21 expressionand/or CCL21 protein relative to a baseline value is indicative of anincreased likelihood that the patient will respond to treatment with thepulmonary hypertension antagonist; wherein the step of assayingcomprises assaying the biological sample for a CCL21 protein or fragmentthereof and wherein the biological sample is selected from blood orplasma or serum.

In yet another embodiment, the method of treating a patient havingpulmonary hypertension, comprising

-   -   a) assaying a biological sample obtained from the patient for        the level of CCL21 expression and/or CCL21 protein; and    -   b) administering a therapeutically effective amount of a        pulmonary hypertension antagonist if the patient has a        statistical significant increased amount of CCL21 expression        and/or CCL21 protein compared to the amount of CCL21 expression        and/or of CCL21 protein to a baseline value that is indicative        of the amount of CCL21 expression and/or of CCL21 protein in a        subject that does not have pulmonary hypertension; wherein the        step of assaying comprises assaying the biological sample for a        CCL21 protein or fragment thereof and wherein the biological        sample is selected from blood or plasma or serum.

In some embodiments, there is provided a method of treating a patienthaving pulmonary hypertension, comprising

-   -   a) assaying a biological sample obtained from the patient for        the level of CCL21 expression and/or CCL21 protein; and    -   b) administering a therapeutically effective amount of a        pulmonary hypertension antagonist if the patient has a        statistical significant increased amount of CCL21 expression        and/or CCL21 protein compared to the amount of CCL21 expression        and/or of CCL21 protein to a baseline value that is indicative        of the amount of CCL21 expression and/or of CCL21 protein in a        subject that does not have pulmonary hypertension and wherein        the pulmonary hypertension antagonist is selected from Calcium        channel blockers, Phosphodiesterase (PDE) 5 inhibitors,        guanylate cyclase (sGC) stimulator, Endothelin receptor        antagonists (ERAs) and Prostacyclin agonists.

Detection (or Assaying) Methods

A variety of methods known or apparent to those skilled in the art maybeemployed to carry out gene or protein expression profiling.

In some embodiments the step of assaying comprises a technique selectedfrom Northern blot analysis, polymerase chain reaction (PCR), reversetranscription-polymerase chain reaction (RT-PCR), TaqMan-based assays,direct sequencing, dynamic allele-specific hybridization, primerextension assays, oligonucleotide ligase assays, temperature gradientgel electrophoresis (TGGE), denaturing high performance liquidchromatography, high-resolution melting analysis, DNA mismatch-bindingprotein assays, capillary electrophoresis, Southern Blot, immunoassays,immunohistochemistry, ELISA, flow cytometry, Western blot, HPLC, andmass spectrometry.

In general, methods of gene expression profiling can be divided into twolarge groups: methods based on hybridization analysis ofpolynucleotides, and other methods based on biochemical detection orsequencing of polynucleotides. The most commonly used methods known inthe art for the quantification of mRNA expression in a sample includenorthern blotting and in situ hybridization (Parker & Barnes, Methods inMolecular Biology 106:247-283 (1999)); RNAse protection assays (Hod,Biotechniques 13:852-854 (1992)); and reverse transcription polymerasechain reaction (RT-PCR) (Weis et al., Trends in Genetics 8:263-264(1992)).

Alternatively, antibodies may be employed that can recognize specificduplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybridduplexes or DNA-protein duplexes. Various methods for determiningexpression of mRNA or protein include, but are not limited to, geneexpression profiling, polymerase chain reaction (PCR) includingquantitative real time PCR (qRT-PCR), microarray analysis that can beperformed by commercially available equipment, following manufacturersprotocols, such as by using the Affymetrix GenChip technology, serialanalysis of gene expression (SAGE) (Velculescu et al., Science270:484-487 (1995); and Velculescu et al., Cell 88:243-51 (1997)),MassARRAY, Gene Expression Analysis by Massively Parallel SignatureSequencing (MPSS) (Brenner et al., Nature Biotechnology 18:630-634(2000)), proteomics, immunohistochemistry (IHC), etc. Preferably mRNA isquantified. Such mRNA analysis is preferably performed using thetechnique of polymerase chain reaction (PCR), or by microarray analysis.Where PCR is employed, a preferred form of PCR is quantitative real timePCR (qRT-PCR).

Immunohistochemistry methods are also suitable for detecting theexpression levels of the biomarker of the present invention. Thus,antibodies or antisera, preferably polyclonal antisera, and mostpreferably monoclonal antibodies specific for each marker are used todetect expression. The antibodies can be detected by direct labeling ofthe antibodies themselves, for example, with radioactive labels,fluorescent labels, hapten labels such as, biotin, or an enzyme such ashorse radish peroxidase or alkaline phosphatase. Alternatively,unlabeled primary antibody is used in conjunction with a labeledsecondary antibody, comprising antisera, polyclonal antisera or amonoclonal antibody specific for the primary antibody.Immunohistochemistry protocols and kits are well known in the art andare commercially available.

Expression levels can also be determined at the protein level, forexample, using various types of immunoassays or proteomics techniques.

In immunoassays, the target diagnostic protein marker is detected byusing an antibody specifically binding to the markers. The antibodytypically will be labeled with a detectable moiety. Numerous labels areavailable which can be generally grouped into the following categories:Radioisotopes, such as 35S, 14C, 1251, 3H, and 1311. The antibody can belabeled with the radioisotope using the techniques described in CurrentProtocols in Immunology, Volumes 1 and 2, Coligen et al. (1991) Ed.Wiley-Interscience, New York, N.Y., Pubs for example and radioactivitycan be measured using scintillation counting.

Fluorescent labels such as rare earth chelates (europium chelates) orfluorescein and its derivatives, rhodamine and its derivatives, dansyl,Lissamine, phycoerythrin and Texas Red are available. The fluorescentlabels can be conjugated to the antibody using the techniques disclosedin “Current Protocols in Immunology”, supra, for example. Fluorescencecan be quantified using a fluorimeter.

Various enzyme-substrate labels are available and U.S. Pat. No.4,275,149 provides a review of some of these. The enzyme generallycatalyzes a chemical alteration of the chromogenic substrate which canbe measured using various techniques. For example, the enzyme maycatalyze a color change in a substrate, which can be measuredspectrophotometrically. Alternatively, the enzyme may alter thefluorescence or chemiluminescence of the substrate. Techniques forquantifying a change in fluorescence are described above. Thechemiluminescent substrate becomes electronically excited by a chemicalreaction and may then emit light which can be measured (using achemiluminometer, for example) or donates energy to a fluorescentacceptor. Examples of enzymatic labels include luciferases (e.g.,firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease,peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase,β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like. Techniques forconjugating enzymes to antibodies are described in O'Sullivan et al.(1981) Methods for the Preparation of Enzyme-Antibody Conjugates for usein Enzyme Immunoassay, in Methods in Enzym. (ed J. Langone & H. VanVunakis), Academic press, New York 73: 147-166.

Examples of enzyme-substrate combinations include, for example:horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate,wherein the hydrogen peroxidase oxidizes a dye precursor (e.g.,orthophenylene diamine (OPD) or 3,3′,5,5′-tetramethyl benzidinehydrochloride (TMB)); alkaline phosphatase (AP) with para-Nitrophenylphosphate as chromogenic substrate; and β-D-galactosidase (β-D-Gal) witha chromogenic substrate (e.g., p-nitrophenyl-β-D-galactosidase) orfluorogenic substrate 4-methylumbelliferyl-β-D-galactosidase.

Numerous other enzyme-substrate combinations are available to thoseskilled in the art. For a general review of these, see U.S. Pat. Nos.4,275,149 and 4,318,980.

In other versions of immunoassay techniques, the antibody need not belabeled, and the presence thereof can be detected using a labeledantibody which binds to the antibody. For the detection of human CCL21protein in plasma and serum samples a custom made immunoassay on theMesoscale Discovery® (MSD) platform was used. MSD'selectrochemiluminescence detection technology uses SULFO-TAG™ labels,which emit light upon electrochemical stimulation initiated at theelectrode surfaces of MULTI-ARRAY and MULTI-SPOT® microplates.

Thus, the diagnostic immunoassays herein may be in any assay format,including, for example, competitive binding assays, direct and indirectsandwich assays such ELISA, and immunoprecipitation assays.

In one embodiment, the method for determining if a subject has pulmonaryhypertension, the method comprises

-   -   a) providing a biological sample obtained from a subject        suspected of having pulmonary hypertension;    -   b) assaying the biological sample for the level of CCL21        expression and/or CCL21 protein; and    -   c) comparing the amount of CCL21 expression and/or of CCL21        protein to a baseline value that is indicative of the amount of        CCL21 expression and/or of CCL21 protein in a subject that does        not have pulmonary hypertension; wherein a statistical        significant increased amount of CCL21 expression and/or of CCL21        protein compared to the baseline value is indicative of        pulmonary hypertension; wherein the step of assaying comprises        assaying the biological sample for a nucleic acid sequence of        CCL21 expression by PCR or RT-PCR; wherein the nucleic acid is        selected from ribonucleic acid (RNA) or a fragment thereof and        complementary deoxyribonucleic acid (cDNA) or a fragment        thereof; and wherein the biological sample is selected from        blood or plasma or serum. Preferably the nucleic acid is cDNA        amplified from CCL21 mRNA.

In another embodiment, the method of predicting the likelihood that apatient having pulmonary hypertension will respond to treatment with apulmonary hypertension antagonist, comprising, assaying a biologicalsample obtained from the patient for the level of CCL21 expressionand/or CCL21 protein; and wherein an increased level of CCL21 expressionand/or CCL21 protein relative to a baseline value is indicative of anincreased likelihood that the patient will respond to treatment with thepulmonary hypertension antagonist; wherein the step of assayingcomprises assaying the biological sample for a nucleic acid sequence ofCCL21 expression by PCR or RT-PCR; wherein the nucleic acid is selectedfrom ribonucleic acid (RNA) or a fragment thereof and complementarydeoxyribonucleic acid (cDNA) or a fragment thereof; and wherein thebiological sample is selected from blood or plasma or serum. Preferablythe nucleic acid is cDNA amplified from CCL21 mRNA.

In yet another embodiment, the method of treating a patient havingpulmonary hypertension, comprising

-   -   a) assaying a biological sample obtained from the patient for        the level of CCL21 expression and/or CCL21 protein; and    -   b) administering a therapeutically effective amount of a        pulmonary hypertension antagonist if the patient has a        statistical significant increased amount of CCL21 expression        and/or CCL21 protein compared to the amount of CCL21 expression        and/or of CCL21 protein to a baseline value that is indicative        of the amount of CCL21 expression and/or of CCL21 protein in a        subject that does not have pulmonary hypertension; wherein the        step of assaying comprises assaying the biological sample for a        nucleic acid sequence of CCL21 expression by PCR or RT-PCR;        wherein the nucleic acid is selected from ribonucleic acid (RNA)        or a fragment thereof and complementary deoxyribonucleic acid        (cDNA) or a fragment thereof; and wherein the biological sample        is selected from blood or plasma or serum. Preferably the        nucleic acid is cDNA amplified from CCL21 mRNA.

In another embodiment, the method for determining if a subject haspulmonary hypertension, the method comprises

-   -   a) providing a biological sample obtained from a subject        suspected of having pulmonary hypertension;    -   b) assaying the biological sample for the level of CCL21        expression and/or CCL21 protein; and    -   c) comparing the amount of CCL21 expression and/or of CCL21        protein to a baseline value that is indicative of the amount of        CCL21 expression and/or of CCL21 protein in a subject that does        not have pulmonary hypertension; wherein a statistical        significant increased amount of CCL21 expression and/or of CCL21        protein compared to the baseline value is indicative of        pulmonary hypertension; wherein the step of assaying comprises        assaying the biological sample for a CCL21 protein or fragment        thereof by immunoassays or ELISA and wherein the biological        sample is selected from blood or plasma or serum.

In another embodiment, the method of predicting the likelihood that apatient having pulmonary hypertension will respond to treatment with apulmonary hypertension antagonist, comprising, assaying a biologicalsample obtained from the patient for the level of CCL21 expressionand/or CCL21 protein by immunoassays or ELISA; and wherein an increasedlevel of CCL21 expression and/or CCL21 protein relative to a baselinevalue is indicative of an increased likelihood that the patient willrespond to treatment with the pulmonary hypertension antagonist; whereinthe step of assaying comprises assaying the biological sample for aCCL21 protein or fragment thereof and wherein the biological sample isselected from blood or plasma or serum.

In yet another embodiment, the method of treating a patient havingpulmonary hypertension, comprising

-   -   a) assaying a biological sample obtained from the patient for        the level of CCL21 expression and/or CCL21 protein; and    -   b) administering a therapeutically effective amount of a        pulmonary hypertension antagonist if the patient has a        statistical significant increased amount of CCL21 expression        and/or CCL21 protein compared to the amount of CCL21 expression        and/or of CCL21 protein to a baseline value that is indicative        of the amount of CCL21 expression and/or of CCL21 protein in a        subject that does not have pulmonary hypertension; wherein the        step of assaying comprises assaying the biological sample for a        CCL21 protein or fragment thereof by immunoassays or ELISA and        wherein the biological sample is selected from blood or plasma        or serum.

Kits of the Invention

The invention provides for a kit for use in determining if a subject haspulmonary hypertension predicting or for use in predicting thelikelihood that a patient having pulmonary hypertension will respond totreatment with a pulmonary hypertension antagonist the kit comprising,

-   -   a) at least one probe capable of detecting the presence of CCL21        expression and/or CCL21 protein; and    -   b) instruction for using the probe to assay a biological sample        from the patient for the presence of CCL21 expression and/or        CCL21 protein.

In one embodiment, the probe is selected from an oligonucleotide thatspecifically hybridizes to a region of a nucleic acid sequence of CCL21expression such as gene-specific or gene-selective probes and/orprimers, for quantitating the expression of CCL21.

The kit may optionally further comprise reagents for the extraction ofRNA from samples, in particular fixed paraffin-embedded tissue samplesand/or reagents for RNA amplification. The kit may comprise containers(including microtiter plates suitable for use in an automatedimplementation of the method), each with one or more of the variousreagents (typically in concentrated form), for example, pre-fabricatedmicroarrays, buffers, the appropriate nucleotide triphosphates (e.g.,dATP, dCTP, dGTP and dTTP; or rATP, rCTP, rGTP and UTP), reversetranscriptase, DNA polymerase, RNA polymerase.

In another embodiment, the probe is a binding molecule capable ofbinding a CCL21 protein or a fragment thereof. Preferably, the bindingmolecule is an antibody or a fragment thereof.

Other binding molecules may be molecules having a scaffold based onfibronectin type III domain (e.g., the tenth module of the fibronectintype III (10 Fn3 domain)), adnectin (Adnectins®), molecules comprisingankyrin derived repeat modules, Affibody® molecules, Anticalins®molecules, Affilin® molecules and protein epitope mimetics.

Examples

This invention is further illustrated by the following examples whichshould not be construed as limiting.

1. Hypoxia/Sugen Rat Model Genechip Profiling

The rat Hypoxia/Sugen model of PH was used to carry out a comparativetranscriptome profiling between rat lung samples with experimental PHand naïve rat lungs.

All animal procedures were conducted in accordance with the British HomeOffice regulations (Scientific Procedures) Act of 1986, UK.

Sugen (250 mg; SU5416; Sigma-Aldrich®) was dissolved in vehicle (12.5ml; 0.5% (wt/vol) carboxyl methylcellulose sodium, 0.9% (wt/vol) NaCl,0.4% (vol/vol) polysorbate, 0.9% (vol/vol) benzyl alcohol in deionizedwater), sonicated for 15 min and then vortexed. On day 0, animals wereanaesthetized, weighed and received Sugen 20 mg/kg by sub-cutaneousinjection. Animals were placed in the hypoxia chamber and the O2 levelwas slowly decreased to 10%. Control animals remained in room air (21%O2) to serve as normoxic controls for the study. After 2 weeks, allanimals were removed from the hypoxia chamber. At week 4, the animalswere subjected to echocardiographic measurements under sevofluoraneanaesthesia and monitored closely until fully recovered. Animalsunderwent RV catheterization for measurement of right ventricularpressure (RVP) under a mixture of ketamin and medetomidine anaesthesia.Following euthanasia by Schedule 1, the left lobe of the lung wasremoved, inflated and fixed in 10% formalin and embedded in paraffin forhistological analysis. Lobes of the right lung were snap frozen fortranscriptional profiling.

Frozen rat lung samples, collected in TT2 tissue bags (K Bioscience®Cat#520021) were crushed using the Covaris CryoPrep CP02. Total RNA fromthe crushed lung samples was extracted by RNeasy Mini kit according tothe manufacturer's protocol (Qiagen™). Genomic DNA was removed bytreatment with DNase I (Turbo DNase kit, Invitrogen). The exactquantification of RNA was determined with a NanoDrop ND-1000spectrophotometer. RNA quality was assessed by analyzing 18S and 28SrRNAs by microfluidics-based electrophoresis on a 2100 Bioanalyzer(Agilent Technologies, Santa Clara, Calif.).

For microarray preparation and analysis, the Affymetrix One-Round InVitro Transcription RNA Amplification Kit was used to amplify 1 μg oftotal RNA. The complementary DNA (cDNA) was synthesised with a primercontaining oligo (dT) and T7 RNA polymerase promoter sequences.Double-stranded cDNA was then purified and used as a template togenerate biotinylated cRNA. The quantity and quality of the amplifiedcRNA was assessed using a NanoDrop ND-1000 Spectrophotometer (ThermoScientific) and an Agilent Bioanalyzer. The biotinylated cRNA wasfragmented and hybridised to Affymetrix Rat GeneChip arrays 230_2. Afterhybridisation, the GeneChip arrays were washed, stained and scannedusing a GeneChip Scanner 3000 7G. Affymetrix GeneChip Operating Softwarewas used for image acquisition. Analysis was performed using GeneSpringGX 11.5.1 software (Agilent Technologies Inc., USA). Data normalisationwas achieved using the Robust Multichip Analysis (RMA) algorithm andbaseline transformation to the median of all samples.

Differentially expressed genes (>1.5 fold, p value 0.05, T-test) thatencode for secreted proteins (likely to be detected in blood samples)and that are associated with remodeling processes were shortlisted forfurther validation (Table 1). Genes associated with remodelling werecollated from the following sources: MetaCore pathway database(http://thomsonreuters.com/metacore formerly GeneGO), Ingenuity PathwayAnalysis database (IPA) (http://www.ingenuity.com/products/ipa), GeneProspector tool in Gene Navigator(http://hugenavigator.net/HuGENavigator). Genes annotated as secreted ordetected in blood were found using the following sources: IngenuityPathway Analysis database (IPA) (http://www.ingenuity.com/products/ipa)and a proprietory data set (SECTRANS).

TABLE 1 Differentially expressed genes in lung samples of Hypoxia Sugentreated animals compared to naïve controls Rat Entrez Fold Rat GeneSymbol Rat Gene Title Gene ID change 1 Cyp1b1 cytochrome P450, family 1,25426 46.4 subfamily b, polypeptide 1 2 Grem1 gremlin 1, cysteine knotsuperfamily, 50566 8.4 homolog (Xenopus laevis) 3 Chia chitinase, acidic113901 4.2 4 Ccl2 chemokine (C-C motif) ligand 2 24770 3.4 5 Serpine1serine (or cysteine) peptidase 24617 3.2 inhibitor, clade E, member 1 6Spp1 secreted phosphoprotein 1 25353 3.1 7 Il1r2 interleukin 1 receptor,type II 117022 3.1 8 Frzb frizzled-related protein 295691 3.0 9 Il6interleukin 6 24498 2.9 10 Cxcl13 chemokine (C-X-C motif) ligand 13498335 2.7 11 Esm1 endothelial cell-specific molecule 1 64536 2.7 12Mmrn1 multimerin 1 500152 2.6 13 Ccl21 chemokine (C-C motif) ligand 21298006 2.6 14 Cthrc1 collagen triple helix repeat 282836 2.6 containing1 15 Plaur plasminogen activator, urokinase 50692 2.5 receptor 16 Tfpi2tissue factor pathway inhibitor 2 286926 2.4 17 C6 complement component6 24237 2.3 18 Dmp1 dentin matrix acidic phosphoprotein 1 25312 2.3 19Ptgs2 prostaglandin-endoperoxide synthase 2 29527 2.3 20 Arhgap1 RhoGTPase activating protein 1 311193 2.3 21 LOC100363145 stabilin 1100363145 2.2 22 Aqp1 aquaporin 1 25240 2.2 23 Fst follistatin 24373 2.124 Reln reelin 24718 2.1 25 Acp5 acid phosphatase 5, tartrate resistant25732 2.1 26 Col18a1 collagen, type XVIII, alpha 1 85251 2.1 27 Lpar6lysophosphatidic acid receptor 6 691774 2.0 28 Nos3 nitric oxidesynthase 3, endothelial 24600 2.0 cell 29 Cxcr4 chemokine (C-X-C motif)receptor 4 60628 1.9 30 Chi3l1 chitinase 3-like 1 89824 1.9 31 Adamts4ADAM metallopeptidase with 66015 1.8 thrombospondin type 1 motif, 4 32Gdf15 growth differentiation factor 15 29455 1.8 33 Tac1 tachykinin 124806 1.8 34 Col1a1 collagen, type I, alpha 1 29393 1.8 35 Angpt2angiopoietin 2 89805 1.8 36 Olr1 oxidized low density lipoprotein 1409141.8 (lectin-like) receptor 1 37 Timp1 TIMP metallopeptidase inhibitor 1116510 1.8 38 Serpine2 serine (or cysteine) peptidase 29366 1.8inhibitor, clade E, member 2 39 Eln elastin 25043 1.8 40 Vcan versican114122 1.7 41 Adora2b adenosine A2B receptor 29316 1.7 42 Cxcl10chemokine (C-X-C motif) ligand 10 245920 1.7 43 Gstp1 glutathioneS-transferase pi 1 24426 1.6 44 Mmp14 matrix metallopeptidase 14 817071.6 (membrane-inserted) 45 Hmox1 heme oxygenase (decycling) 1 24451 1.646 Ctsk cathepsin K 29175 1.6 47 Il1r1 interleukin 1 receptor, type I25663 1.6 48 Pthlh parathyroid hormone-like hormone 24695 1.6 49 Axl Axlreceptor tyrosine kinase 308444 1.6 50 Gch1 GTP cyclohydrolase 1 292441.6 51 Inhba inhibin beta-A 29200 1.5 52 Cxcl12 chemokine (C-X-C motif)ligand 12 24772 1.5 (stromal cell-derived factor 1) 53 Hp haptoglobin24464 1.5 54 Fn1 fibronectin 1 25661 1.5 55 Il6st interleukin 6 signaltransducer 25205 1.5 56 Il1rn interleukin 1 receptor antagonist 605821.5 57 Des desmin 64362 1.5 58 Vegfa vascular endothelial growth factorA 83785 −1.5 59 Ace angiotensin I converting enzyme 24310 −1.6(peptidyl-dipeptidase A) 12. Assessment of Candidate Biomarker mRNA Levels in the Hypoxia SugenRat Model of Idiopathic Pulmonary Arterial Hypertension FollowingTreatment with Imatinib

All animal procedures were conducted as described above.

In this study, following administration of an initial dose of Sugen onday 0 and a period of 14 days in a hypoxia chamber following, rats wereadministered 100 mg/kg of Imatinib or vehicle control daily for afurther two weeks.

Following euthanasia by Schedule 1, the left lobe of the lung wasremoved, inflated and fixed in 10% formalin and embedded in paraffin forhistological analysis, by airway inflation. Tissue sections (3 μm) werestained with antibodies against von Willebrand factor (vWF) and α-smoothmuscle actin (α-SMA). Slides were examined using a DMLB and confocalmicroscope, digital camera, and 1M50 imaging software (LeicaMicrosystems, London, UK). Small pulmonary vessels (10-100 μm diameterindicated by vWF staining) were assessed for degrees of circumferentialα-SMA-positive staining indicative of muscularisation. Lobes of theright lung were snap frozen for candidate biomarker mRNA expressionmeasurements. Total RNA was extracted and underwent quality control asmentioned in section above.

cDNA was synthesised using the High Capacity RNA-to-cDNA Kit(Invitrogen) according to the kit manufacturer's protocol. QPCR wasperformed on an ABI Prism 7900HT sequence detection system (AppliedBiosystems, USA), using TaqMan Universal PCR Master Mix (AppliedBiosystems). Taqman assays were purchased from Applied Biosystems®.Relative expression was normalised to a combination of 10 differenthousekeeping genes. Data were analyzed using the SDS RQ Manager,software (Applied Biosystems, version 2.4). Normalised gene expressionvalues for each gene (2^(−Δct)) were plotted and analysed using atwo-way ANOVA in Graph Pad Prism 6.02.

The expression of all transcripts listed on Table 1 was assessed in lungsamples from animals treated with vehicle or Imatinib. The following 6gene transcripts were found to be upregulated in rat Hy/Su lungs anddown-regulated with treatment with Imatinib: Ccl21, Col18a1, Cxcl12,Cxcl13, Dmp1, Frzb (FIG. 1). In addition, we measured CCL21 mRNAexpression levels in lung samples from groups treated with imatinib orvehicle on day 28 and control naïve animals and observed an increase ofCCL21 mRNA levels in lungs of vehicle treated animals compared to naïvecontrols and a decrease following treatment with Imatinib (FIG. 3, uppergraphs). CCL21 expression levels in lung samples from these groupscorrelated significantly with both right ventricular pressure andarterial muscularisation (P<0.0001)

We therefore concluded that transcript levels of the above genes aredown-regulated in response to an anti-remodelling agent in the lungs ofanimals with pulmonary hypertension as a result of the therapeutic drugadministration.

3. Assessment of Candidate Biomarker mRNA Correlation with VascularRemodelling Readouts in a Rat Hypoxia/Sugen Longitudinal

All animal procedures were conducted as described above. In this studyfollowing administration of an initial dose of Sugen on day 0 and aperiod of 21 days in a hypoxia chamber, rats developed elevated rightventricular pressure and arterial muscularisation. Following euthanasiaby Schedule 1, the left lobe of the lung was removed for histologicalanalysis and the right lobe for mRNA analysis. We measured all 6candidate biomarker mRNA expression levels in lung samples from thefollowing study timepoints: weeks 3, 5, 8 and 14 and naïve animals (n=6in each group). Pearson correlation was used to assess the significanceof the correlation between candidate biomarker mRNA expression andpercentage of muscularisation, right ventricular pressure (RVP) andnumber of occluded vessels. Transcript expression of all markersassessed in lung samples from these groups correlated significantly withat least two of the three vascular remodeling readouts: percentage ofarterial muscularisation, percentage of occluded vessel and rightventricular pressure (Table 2). We therefore concluded that transcriptsof the candidate biomarker assessed are indicative of the degree ofvascular remodeling.

TABLE 2 Correlations of candidate biomarker transcript expression levelswith vascular remodeling readouts. R value P value Transcriptmuscularisation muscularisation CCL21 0.7102 <0.0001 Cxcl12 0.626<0.0001 Frzb 0.6292 <0.0001 Cxcl13 0.6164 <0.0001 Col18a1 0.4072 0.0152Dmp1 0.3933 0.0194 R value lumen P value lumen Transcript occlusionocclusion CCL21 0.7344 <0.0001 Cxcl12 0.5331 0.0008 Frzb 0.4921 0.0023Cxcl13 0.3482 0.0374 Col18a1 0.347 0.0381 Dmp1 0.1549 0.3671 R value Rvalue Transcript RVP RVP CCL21 0.7164 <0.0001 Cxcl12 0.6611 <0.0001 Frzb0.7078 <0.0001 Cxcl13 0.7183 <0.0001 Col18a1 0.4877 0.0091 Dmp1 0.47960.0031

4. Assessment of Candidate Biomarker Protein Levels in Serum and PlasmaSamples of PH Patients and Matched Controls

To determine whether circulating protein levels of candidate biomarkersare elevated in serum and plasma PH patient samples compare to matchedcontrols, we developed immunoassays for CCL21, CXCL12, CXCL13 andCol181a and measured circulating levels of these in 30 PH patients and25 age, ethnicity and gender ratio matched controls.

Human peripheral blood samples were obtained and handled in accordancewith an approved Ethical Review Board application. Matched serum andplasma samples from 30 pulmonary hypertension patients were collected.Out of the 30 PH patients, 24.8% belonged to Group 1, 28.4% to Group 2,10% to Groups 2 and 3, 33.3% to Group 3 and 3.5% to Group 4 according tothe World Health Organisation (WHO) classification system (Dana Point2008)²

Immunoassays using MSD Coated Custom plates were carried out as permanufacturer's recommendations. Briefly, plates were incubated withproprietary Diluent 2 at 25 μl/well, sealed with adhesive cover film andincubated for 30 minutes at room temperature on a plate shaker (300-1000rpm). CCL21 Recombinant protein (R&D Systems®, cat# DY366, Part 841709)was reconstituted 1% BSA (Gibco, cat#15260-037)/PBS (Gibco, cat#14190)and was added at 10,000 pg/ml and 1:5 serial dilutions were performedwith Diluent 2, with the 8^(th) point as the zero standard (0 pg/ml).

Standards, samples, and assay controls were added at 25 μl/well to MSDplate with Diluent 2. Plate was sealed and incubated at room temperaturefor 2 hours on a plate shaker (300-1000 rpm). The wells were then washedthree times with wash buffer (0.05% Tween-20 in PBS pH7.4 Sigma™, cat#P3563-10PAK).

CCL21 detection and capture antibodies (Human CCL21/6Ckine DuoSet ELISAdevelopment system, R&D Systems®, cat# DY366, Parts 841707 and 841708,supplied with MSD Coated Custom Plate) were reconstituted according tothe R&D system DuoSet protocol, at a final concentration of 1 μg/ml.Detection antibody solution was added to the washed plate. The plate wassealed and incubated for 2 hours with shaking at room temperature. Aftera final wash step reverse pipetting was used to add 150 μl of 2× ReadBuffer T (diluted with an equal volume of H₂O) and the plate was readusing an MSD instrument SECTOR Imager 6000.

For the statistical analysis, an unpaired-t test was performed usingGraphPad prism 6. A Receiver Operating Characteristic (ROC) curveanalysis was performed using GraphPad Prism 6. The area under the curve(AUC), as generated by the software, reflects the specificity and theselectivity of the biomarker/. An AUC of 1 indicates a biomarker 100%sensitive and specific in discriminating two populations.

We found that CCL21, but not Col18a1, CXCL12 or 13 (data not shown), wasup-regulated in PH patients compared to matched controls in both serumand plasma samples (FIG. 2). Receiver Operating Characteristics (ROC)curve analysis of the data sets indicated that CCL21 circulating levelsare able to discriminate patients from controls with high sensitivityand specificity, with an are under the curve (AUC) of 0.91 in serumsamples and 0.89 in plasma samples (FIG. 2). We therefore concluded thatCCL21 is upregulated in serum and plasma samples of PH patients comparedto matched controls and is able to discriminate patients from controlswith high sensitivity/specificity

5. Human IHC CCL21 Protein Data from PH Patients

Formalin fixed paraffin embedded tissue sections from PH were sourcedfrom the University of Cambridge from patients undergoing lungtransplantation under an approved informed consent and institutionalagreement.

CCL21 was detected by immunohistochemistry on a Ventana Discovery XTusing the following protocol. Briefly, sections were dewaxed usingEZprep solution, and high pH8 antigen retrieval was performed using theVentana cc1 reagent. CCL21 was detected using goat anti-human CCL21antibody (R&D System® AF366, 3.33 ug/ml)—the antibody was incubated for12 hours at room temperature. Secondary antibody was biotinylated rabbitanti-goat (DAKO E0466) diluted to 1/200, 20 minute incubation at 37° C.Biotinylated secondary antibody was detected using the DABMap kit.(Ventana 650-010). Sections were counterstained using Harris'haematoxylin and coverslipped. Images were scanned using the Aperio XTslide scanner and analysed using Definiens Tissue Studio.

CCL21 protein was detected in PAH lesions (FIG. 3(A-D)) from advanced PHpatients in areas with subepithelial/epithelial and alveolar macrophagesas well as lymphatic vessels. We therefore concluded that CCL21 isexpressed at the site of the disease pathology.

REFERENCES

-   1. Schermuly, R. T., Ghofrani, H. A., Wilkins, M. R. &    Grimminger, F. Mechanisms of disease: pulmonary arterial    hypertension. Nature reviews. Cardiology 8, 443-455 (2011).-   2. Galie, N. et al. Guidelines for the diagnosis and treatment of    pulmonary hypertension. The European respiratory journal 34,    1219-1263 (2009).-   3. Dewachter, L. et al. Angiopoietin/Tie2 pathway influences smooth    muscle hyperplasia in idiopathic pulmonary hypertension. American    journal of respiratory and critical care medicine 174, 1025-1033    (2006).-   4. Fike, C. D., Slaughter, J. C., Kaplowitz, M. R., Zhang, Y. &    Aschner, J. L. Reactive oxygen species from NADPH oxidase contribute    to altered pulmonary vascular responses in piglets with chronic    hypoxia-induced pulmonary hypertension. American journal of    physiology. Lung cellular and molecular physiology 295, L881-888    (2008).-   5. Price, L. C. et al. Inflammation in pulmonary arterial    hypertension. Chest 141, 210-221 (2012).-   6. Rabinovitch, M. Molecular pathogenesis of pulmonary arterial    hypertension. The Journal of clinical investigation 122, 4306-4313    (2012).-   7. Voelkel, N. F., Gomez-Arroyo, J., Abbate, A., Bogaard, H. J. &    Nicolls, M. R.

Pathobiology of pulmonary arterial hypertension and right ventricularfailure. The European respiratory journal 40, 1555-1565 (2012).

-   8. Kherbeck, N. et al. The role of inflammation and autoimmunity in    the pathophysiology of pulmonary arterial hypertension. Clinical    reviews in allergy & immunology 44, 31-38 (2013).-   9. Perros, F. et al. Fractalkine-induced smooth muscle cell    proliferation in pulmonary hypertension. The European respiratory    journal 29, 937-943 (2007).-   10. Humbert, M. et al. Increased interleukin-1 and interleukin-6    serum concentrations in severe primary pulmonary hypertension.    American journal of respiratory and critical care medicine 151,    1628-1631 (1995).-   11. Soon, E. et al. Elevated levels of inflammatory cytokines    predict survival in idiopathic and familial pulmonary arterial    hypertension. Circulation 122, 920-927 (2010).-   12. Tamby, M. C. et al. Anti-endothelial cell antibodies in    idiopathic and systemic sclerosis associated pulmonary arterial    hypertension. Thorax 60, 765-772 (2005).-   13. Terrier, B. et al. Identification of target antigens of    antifibroblast antibodies in pulmonary arterial hypertension.    American journal of respiratory and critical care medicine 177,    1128-1134 (2008).-   14. Tuder, R. M., Groves, B., Badesch, D. B. & Voelkel, N. F.    Exuberant endothelial cell growth and elements of inflammation are    present in plexiform lesions of pulmonary hypertension. The American    journal of pathology 144, 275-285 (1994).-   15. Carragher, D. M., Rangel-Moreno, J. & Randall, T. D. Ectopic    lymphoid tissues and local immunity. Seminars in immunology 20,    26-42 (2008).-   16. Brusselle, G. G., Demoor, T., Bracke, K. R., Brandsma, C. A. &    Timens, W. Lymphoid follicles in (very) severe COPD: beneficial or    harmful? The European respiratory journal 34, 219-230 (2009).-   17. Marchal-Somme, J. et al. Cutting edge: nonproliferating mature    immune cells form a novel type of organized lymphoid structure in    idiopathic pulmonary fibrosis. J Immunol 176, 5735-5739 (2006).-   18. Sato, M. et al. The role of intrapulmonary de novo lymphoid    tissue in obliterative bronchiolitis after lung transplantation. J    Immunol 182, 7307-7316 (2009).-   19. Perros, F. et al. Pulmonary lymphoid neogenesis in idiopathic    pulmonary arterial hypertension. American journal of respiratory and    critical care medicine 185, 311-321 (2012).-   20. Aloisi, F. & Pujol-Borrell, R. Lymphoid neogenesis in chronic    inflammatory diseases. Nature reviews. Immunology 6, 205-217 (2006).-   21. Nagaya, N. et al. Plasma brain natriuretic peptide as a    prognostic indicator in patients with primary pulmonary    hypertension. Circulation 102, 865-870 (2000).-   22. Giannakoulas, G. et al. Usefulness of natriuretic Peptide levels    to predict mortality in adults with congenital heart disease. The    American journal of cardiology 105, 869-873 (2010).-   23. Galie, N. et al. Ambrisentan for the treatment of pulmonary    arterial hypertension: results of the ambrisentan in pulmonary    arterial hypertension, randomized, double-blind, placebo-controlled,    multicenter, efficacy (ARIES) study 1 and 2. Circulation 117,    3010-3019 (2008).-   24. Mauritz, G. J. et al. Usefulness of serial N-terminal pro-B-type    natriuretic peptide measurements for determining prognosis in    patients with pulmonary arterial hypertension. The American journal    of cardiology 108, 1645-1650 (2011).-   25. Yoshida, R. et al. Secondary lymphoid-tissue chemokine is a    functional ligand for the CC chemokine receptor CCR7. The Journal of    biological chemistry 273, 7118-7122 (1998).

What is claimed is:
 1. A method for determining if a subject haspulmonary hypertension, comprising a) providing a biological sampleobtained from a subject suspected of having pulmonary hypertension; b)assaying the biological sample for the level of CCL21 expression and/orCCL21 protein; and c) comparing the amount of CCL21 expression and/or ofCCL21 protein to a baseline value that is indicative of the amount ofCCL21 expression and/or of CCL21 protein in a subject that does not havepulmonary hypertension; wherein a statistical significant increasedamount of CCL21 expression and/or of CCL21 protein compared to thebaseline value is indicative of pulmonary hypertension.
 2. A method ofpredicting the likelihood that a patient having pulmonary hypertensionwill respond to treatment with a pulmonary hypertension antagonist,comprising, assaying a biological sample obtained from the patient forthe level of CCL21 expression and/or CCL21 protein; and wherein anincreased level of CCL21 expression and/or CCL21 protein relative to abaseline value is indicative of an increased likelihood that the patientwill respond to treatment with the pulmonary hypertension antagonist. 3.A method of treating a patient having pulmonary hypertension, comprisinga) assaying a biological sample obtained from the patient for the levelof CCL21 expression and/or CCL21 protein; and b) administering atherapeutically effective amount of a pulmonary hypertension antagonistif the patient has a statistical significant increased amount of CCL21expression and/or CCL21 protein compared to the amount of CCL21expression and/or of CCL21 protein to a baseline value that isindicative of the amount of CCL21 expression and/or of CCL21 protein ina subject that does not have pulmonary hypertension.
 4. The methodaccording to any one of claims 1 to 3, wherein the step of assayingcomprises assaying the biological sample for a nucleic acid sequence ofCCL21 expression.
 5. The method according to claim 4, wherein thenucleic acid is selected from CCL21 ribonucleic acid (RNA) or a fragmentthereof and complementary deoxyribonucleic acid (cDNA) or a fragmentthereof.
 6. The method according to any one of claims 1 to 3, whereinthe step of assaying comprises assaying the biological sample for aCCL21 protein or fragment thereof.
 7. The method according to any one ofclaims 1 to 6, wherein the biological sample is selected from blood,serum, plasma, urine, saliva, faeces and a tissue sample.
 8. The methodaccording to any one of claims 1 to 7, wherein the step of assayingcomprises a technique selected from Northern blot analysis, polymerasechain reaction (PCR), reverse transcription-polymerase chain reaction(RT-PCR), TaqMan-based assays, direct sequencing, dynamicallele-specific hybridization, primer extension assays, oligonucleotideligase assays, temperature gradient gel electrophoresis (TGGE),denaturing high performance liquid chromatography, high-resolutionmelting analysis, DNA mismatch-binding protein assays, capillaryelectrophoresis, Southern Blot, immunoassays, immunohistochemistry,ELISA, flow cytometry, Western blot, HPLC, and mass spectrometry.
 9. Akit for use in determining if a subject has pulmonary hypertensionpredicting or for use in predicting the likelihood that a patient havingpulmonary hypertension will respond to treatment with a pulmonaryhypertension antagonist, the kit comprising, a) at least one probecapable of detecting the presence of CCL21 expression and/or CCL21protein; and b) instruction for using the probe to assay a biologicalsample from the patient for the presence of CCL21 expression and/orCCL21 protein.
 10. The kit according to claim 9, wherein the probe isselected from an oligonucleotide that specifically hybridizes to aregion of a nucleic acid sequence of CCL21 expression or bindingmolecule capable of binding a CCL21 protein or a fragment thereof. 11.The kit according to claim 9 wherein the binding molecule is an antibodyor a fragment thereof.